Preparation and Characterization of New Liposomes. Bactericidal Activity of Cefepime Encapsulated into Cationic Liposomes

Abstract

Cefepime is an antibiotic with a broad spectrum of antimicrobial activity. However, this antibiotic has several side effects and a high degradation rate. For this reason, the preparation and characterization of new liposomes that are able to encapsulate this antibiotic seem to be an important research line in the pharmaceutical industry. Anionic and cationic liposomes were prepared and characterized. All cationic structures contained the same cationic surfactant, N,N,N-triethyl-N-(12-naphthoxydodecyl)ammonium. Results showed a better encapsulation-efficiency percentage (EE%) of cefepime in liposomes with phosphatidylcholine and cholesterol than with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The presence of cholesterol and the quantity of egg-yolk phospholipid in the liposome increased the encapsulation percentage. The bactericidal activity against Escherichia coli of cefepime loaded into liposomes with phosphatidylcholine was measured. The inhibitory zone in an agar plate for free cefepime was similar to that obtained for loaded cefepime. The growth-rate constant of E. coli culture was also measured in working conditions. The liposome without any antibiotic exerted no influence in such a rate constant. All obtained results suggest that PC:CH:12NBr liposomes are biocompatible nanocarriers of cefepime that can be used in bacterial infections against Escherichia coli with high inhibitory activity.

Keywords: bactericidal activity; cefepime; encapsulation; liposome; surfactant.; zeta potential.

Figure 1

Cefepime structure.

Figure 2

Structure of lipids and cholesterol used in this work.

Figure 3

Intensity/size distribution of a liposome containing l-α-phosphatidylcholine (PC), cholesterol (CH), and N,N,N-triethyl-N-(12-naphthoxy dodecyl)ammonium bromide (12NBr) (Sample C in Table 1) in the (A) absence and (B) presence of cefepime.

Figure 4

Dependence of liposome size on the α parameter for different liposomes. Error bars represent standard deviation in each α value (n = 5).

Figure 5

Absorbance dependence on time in a dialysis process (liposome PC + CH, Sample A). Error bars represent standard deviation in each time value (n = 5).

Figure 6

(A) Release of cefepime from liposome PC:CH:12NBr (Sample C) at 37 °C. (B) Size and Pdi of liposome PC:CH:12NBr (Sample C) at different times.

Figure 7

Zones of inhibition produced by cefepime after 24 h of incubation at different antibiotic concentrations and 37 °C: (A) [Cefepime]_free = 120 µg/mL (1); [Cefepime]_encapsulated = 120 µg/mL (2), 50 µg/mL (5), 20 µg/mL (6). Disks 3 and 4 only contain liposomes. Sample D in Table 1 was used in this assay. (B) [Cefepime]_free = 70 µg/mL (1); [Cefepime]_encapsulated = 120 µg/mL (3), 70 µg/mL (4). Disk 2 contains liposome without cefepime. Sample F in Table 1 was used in this assay.

Figure 8

Dependence of inhibition zone with time in the presence and absence of PC:CH:12NBr liposomes at 37 °C. Error bars represent standard deviation in each time value (n = 3).

Figure 9

Growth inhibition of E. coli in the absence and presence of cefepime (free and encapsulated in PC:CH:12NBr liposomes) at different antibiotic concentrations.

Figure 10

Determination of growth-rate constant for E. coli by using Equation (4).